Constitutive patterns of gene expression regulated by RNA-binding proteins

Background

RNA-binding proteins regulate a number of cellular processes, including synthesis, folding, translocation, assembly and clearance of RNAs. Recent studies have reported that an unexpectedly large number of proteins are able to interact with RNA, but the partners of many RNA-binding proteins are still uncharacterized.

Results

We combined prediction of ribonucleoprotein interactions, based on catRAPID calculations, with analysis of protein and RNA expression profiles from human tissues. We found strong interaction propensities for both positively and negatively correlated expression patterns. Our integration of in silico and ex vivo data unraveled two major types of protein–RNA interactions, with positively correlated patterns related to cell cycle control and negatively correlated patterns related to survival, growth and differentiation. To facilitate the investigation of protein–RNA interactions and expression networks, we developed the catRAPID express web server.

Conclusions

Our analysis sheds light on the role of RNA-binding proteins in regulating proliferation and differentiation processes, and we provide a data exploration tool to aid future experimental studies.     

Soluble proteins from chemosensory organs of Eurycantha calcarata (Insects, Phasmatodea)

Three related nucleotide sequences, encoding mature proteins of 108-113 amino acids, have been obtained from antennal cDNA of the Phasmid Eurycantha calcarata. Among these, one is also expressed in the tarsi as demonstrated by N-terminal sequence and mass spectrometric analyses of protein samples isolated from both organs. PCR experiments performed with specific primers, showed that this species is also expressed in the mouth organs and in the cuticle, while the other two are antennal specific. All three isoforms are similar to Drosophila OS-D and other proteins reported in several insect orders, but one of them is significantly different from the other two. The best conserved elements are the N-terminal region and the four cysteine residues. Accurate ESMS measurements indicated that all cysteines are involved in two disulphide bonds and ruled out the occurrence of additional post-translational modifications. Polyclonal antibodies, raised against the purified protein, did not react with proteins of the same class expressed in another Phasmid species, Carausius morosus, and in the orthopteran Schistocerca gregaria, nor did antibodies against these proteins recognise those of E. calcarata.     

Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.     

Carbon isotope composition as a tool to control the quality of herbs and medicinal plants

Isotope screening is a simple test for determining the photosynthetic pathway used by plants. The scope of this work was to classify the photosynthetic type of some herbs and medicinal plants through studies of the carbon isotope composition (δ¹³C). Also, we propose the use of carbon isotope composition as a tool to control the quality of herbs and medicinal plants. For studies of δ¹³C, δ¹³C‰ = [R (sample)/R (standard) − 1] × 10⁻³, dry leaves powdered in cryogenic mill were analyzed in a mass spectrometer coupled with an elemental analyzer for determining the ratio R = ¹³CO₂/¹²CO₂. In investigation of δ¹³C of 55 species, 23 botanical families, and 44 species possessed a C₃ photosynthetic type. Six species found among the botanical families Euphorbiaceae and Poaceae were C₄ plants, and 5 species found among the botanical families Agavaceae, Euphorbiaceae, and Liliaceae possessed CAM-type photosynthesis. Carbon isotope composition of plants can be used as quality control of herbs and medicinal plants, allowing the identification of frauds or contaminations. Also, the information about the photosynthetic type found for these plants can help in introducing and cultivating exotic and wild herbs and medicinal plants.     

Chlorophyll fluorescence and chlorophyll content in field-grown potato as affected by nitrogen supply, genotype, and plant age

Field experiments were conducted in Sicily (south Italy) to assess chlorophyll (Chl) fluorescence parameters in response of potato crop to nitrogen dose, to variation in genotype and in plant age, and to detect relationships between Chl content, fluorescence parameter F_v/F_m, and tuber yield. The experiment included five nitrogen doses (0, 10, 20, 30, and 40 g m⁻²) and four genotypes (Spunta, Sieglinde, Daytona, and Igea). Chl fluorescence parameters (initial fluorescence, F₀, maximum fluorescence, F_m, variable fluorescence, F_v, F_v/F_m, T_max (the time required to reach F_m), and Chl content were measured weekly between the appearance of the fifth and sixth leaves and the onset of plant senescence. A positive linear relationship was established between nitrogen supply and Chl content, F₀, and T_max. Nitrogen supply up to 10 g m⁻² also had a positive effect on F_m and F_v, but above this rate it reduced F_v/F_m. Spunta had the highest Chl content, F_m, F_v, and F_v/F_m, but the lowest F₀, whereas Sieglinde had the lowest Chl content, F_v, F_v/F_m, and T_max and the highest F₀. The cvs. Igea and Daytona exhibited intermediate Chl fluorescence parameters. Chl content and T_max decreased with increasing plant age, whereas F₀, F_m, and F_v increased until complete canopy development and thereafter declined until crop maturity. Tuber and plant dry matter yield were significantly correlated with Chl content, F₀, and T_max. Thus Chl fluorescence and content detect differences in the response of potato to N supply, can discriminate between genotypes, predict plant age, and yield performance under field conditions.     

Development of “universal” gene-specific markers from Malus spp. cDNA sequences, their mapping and use in synteny studies within Rosaceae

The Rosaceae contains many economically valuable crop genera, including Malus (apple), Fragaria (strawberry), and Prunus (stone fruit). There has been increasing interest in the development of linkage maps for these species, with a view to marker-assisted selection to assist breeding programs and, recently, in the development of transferable markers to permit syntenic comparisons of maps of different rosaceous genera. In this investigation, a set of Malus cDNA sequences were downloaded from the European Molecular Biology Laboratory database. The sequences were aligned with homologous full-length Arabidopsis genomic DNA sequences to identify putative intron–exon junctions and conserved flanking exon sequences. Primer pairs were designed from the conserved exon sequences flanking predicted intron–exon junctions in the Malus cDNA sequences. These were used to amplify products by polymerase chain reaction from the parents of the Malus mapping progeny “Fiesta” × “Totem.” Eleven loci, representing ten genes (39%), were polymorphic in the “Fiesta” × “Totem” population and mapped to seven Malus linkage groups. Transferability to other rosaceous genera was high, with primer pairs representing 85% of genes, amplifying products from Fragaria and primer pairs representing 85% of genes, amplifying products from Prunus genomic DNA. These primers were screened in the Fragaria and Prunus mapping bin sets and 38% of the genes were successfully located on both maps. Analysis of the markers mapped in more than one rosaceous genus revealed patterns of synteny between genera, while a comparison with the physical positions of homologous genes on the Arabidopsis genome revealed high sequence conservation but only fragmentary patterns of macrosynteny.     

The shape modulation of osteoblast–osteocyte transformation and its correlation with the fibrillar organization in secondary osteons

Cortex fractured surface and graded osmic maceration techniques were used to study the secretory activity of osteoblasts, the transformation of osteoblast to osteocytes, and the structural organization of the matrix around the cells with scanning electron microscopy (SEM). A specialized membrane differentiation at the base of the cell was observed with finger-like, flattened processes which formed a diffuse meshwork. These findings suggested that this membrane differentiation below the cells had not only functioned in transporting collagen through the membrane but also in orienting the fibrils once assembled. Thin ramifications arose from the large and flat membrane foldings oriented perpendicular to the plane of the osteoblasts. This meshwork of fine filaments could not be visualized with SEM because they were obscured within the matrix substance. Their 3-D structure, however, should be similar to the canalicular system. The meshwork of large, flattened processes was no more evident in the cells which had completed their transformation into osteocytes.     

Genetic relationships, structure and parentage simulation among the olive tree (Olea europaea L. subsp. europaea) cultivated in Southern Italy revealed by SSR markers

In this work, we assess both the morphological and genetic diversity of 68 important olive cultivars from three Southern Italian regions: Calabria, Campania and Sicily. Twenty-five phenotypic traits were evaluated and 12 simple sequence repeat (SSR) markers were analysed. All SSR primers were polymorphic and reliable. The total number of alleles per locus varied from 5 to 19 with an average number of 13.1 and a mean polymorphic information content (PIC) of 0.81. These results suggested high genetic diversity within these three olive germplasm collections. Morphological traits also showed significant variability amongst cultivars. Two cases of identity were found and ten statistically significant cases of putative parent/sibling were discovered by performing a SSR-based parentage simulation analysis with CERVUS. The Mantel test indicated low but significant correlations between the morphological data and SSR allelic frequency, origin and SSR allelic frequency, and origin and morphology. Structure software allowed inference of relationships between the three olive germplasm collections and allowed us to obtain the most consistent grouping and to identify putative admixed or exchanged cultivars. Cluster and multivariate analysis, based on morphological traits, revealed geographic grouping in agreement with UPGMA dendrogram and structure analysis using SSRs. Sicilian cultivars showed a more homogenous genetic makeup, probably due to geographical isolation, whilst Calabrian and Campanian cultivars seemed to have a less distinct genetic structure, with a greater degree of intermixing. A correlation between the presence of certain SSR alleles and fruit size was also found.